Fig. 1Lhx5 promotes forebrain development. In this and subsequent figures, the probes used for whole-mount in situ hybridization are listed in the upper right corner of each panel. Genotypes or experimental manipulations are indicated in the lower left corners. Developmental stages are indicated in the lower right corners. Unless otherwise noted, gastrula stage embryos are orientated in animal pole view, rostral to the top; post-gastrulation stage embryos in lateral view, rostral to the top and dorsal to the right. ctl, control embryos. (A-D) lhx5 is expressed in rostral regions during embryonic development. (A,B) Dorsal to the right, lateral (A) and animal pole (B) views. (D) A gap can be seen between the lhx5 and pax2a expression domains. (E-N) lhx5 gain of function causes expansion of the forebrain. (E,F) Forebrain boundaries are marked by broken lines. (G,H) Dorsal view, bud stage, pax6a expression. (I-N) Dorsal view, rostral to the top. Embryos were dissected and flat mounted in glycerol after in situ hybridization. Partial overlays of panels were made with PhotoShop (Adobe) and brightness in the overlapped regions was adjusted so that the backgrounds match. (O-V) Inhibition of Lhx5 function compromises forebrain development. (U,V) Red arrowheads mark the forebrain-midbrain boundary. (W-Z) lhx5 morpholino knockdown alters pax6a and pax2a expression. Embryos were dissected and flat mounted in glycerol after in situ hybridization. Dorsal view, rostral to the top. Scale bar in Z: 250 μm for A-H,Q-V; 200 μm for I-K; 167 μm for L-N; 150 μm for O,P; 100 μm for W-Z.

Fig. S2Lhx5, Sfrp1a and Sfrp5 regulate the size of the forebrain. Quantitative measurement of forebrain sizes. (A,B) An uninjected control embryo (A) and an lhx5 mRNA-injected embryo (B) are shown to illustrate how quantitative measurements are carried out. Injected embryos and uninjected control embryos were fixed and processed for in situ hybridization with ptc1 probes. Images of labeled embryos were analyzed with Axiovision software (Carl Zeiss). Two parameters were measured for each image. Green double arrow (head thickness) crosses the apex of forebrain and is approximately perpendicular to the tangent plane at the apex. Forebrain length was defined as the distance between the anterior limit and the diencephalic bending point of ptc1 labeling (red stars). The ptc1 bending point is located at the zona limitans intrathalamica that separates the anterior diencephalon and the posterior diencephalon. (C,D) Measurements were normalized by dividing by the values of uninjected controls and multiplying by 100. ** denotes significant differences between uninjected control and affected injected embryos (t-test, P<0.01). For each measurement, the sample size was n=10.

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